CN107096037A - A kind of method that enzymatic small molecule self assembly prepares nanogel - Google Patents
A kind of method that enzymatic small molecule self assembly prepares nanogel Download PDFInfo
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Abstract
The present invention relates to a kind of method that enzymatic small molecule self assembly prepares nanogel, this method is first to carry out amination modification, carboxylated modification to the surface of nano particle successively, reacted afterwards by acid amides by enzyme modification in nano grain surface, it is eventually adding small-molecular peptides, in the presence of enzyme, small-molecular peptides are converted into gelator, and are self-assembly of nanogel in nano grain surface.Compared with prior art, the present invention enters small-molecular peptides by enzymatic and is converted into gelator, and preferentially it is assembled into small molecule gel in nano grain surface, and then prepare nanogel, the nanogel has bioactivity and multifunctionality, can as growth factor, medicine and cell carrier, the treatment etc. repaired applied to cell culture, wound provides new thinking for the preparation of nanogel.
Description
Technical field
The invention belongs to nano meter biomaterial technical field, it is related to a kind of enzymatic small molecule self assembly and prepares nanogel
Method.
Background technology
Hydrogel is the gel using water as decentralized medium, and one is introduced in the water soluble polymer with cross-linked network
Part hydrophobic grouping and hydrophilic residue, hydrophilic residue and water molecules, netted inside is connected to by hydrone, and hydrophobic residue
Water-swellable.
With continuing to develop for hydrogel techniques, the research of hydrogel is progressively from bulk gels, to microgel, then to nanometer
Gel.Nanogel is that a class is micro- by the hydrogel of physics or the nanoscale with three-dimensional net structure of chemical crosslinking
Grain, is generally polymerized, diameter is in below 200nm by high polymer monomer.Nanogel for other gels, with
Lower advantage：First, size is small, is easily phagocytized by cells；2nd, the various diaphragms in human body are penetrated readily through, such as meninx, so as to
Realize that brain is administered；3rd, carrier medicine carrying efficiency is high.Therefore, nanogel receives people and more and more paid close attention in recent years.
At present, the preparation method of nanogel is mainly blending method, i.e., by inorganic nano-particle and polymer according to difference
Process combining form, be to prepare a kind of simplest method of polymer nanocomposite composite aquogel at present, the advantage of this method is
The preparation of nano-particle and polymer is separately carried out, and can effectively control the form and size of nano-particle, but when having the disadvantage blending
Nano-particle is easily reunited, and is difficult to disperse.
Enzyme-catalyzed polymerization reaction be one of focus of Recent study, in terms of hydrogel is prepared, due to enzyme have efficiently,
Single-minded, stable the characteristics of so that it, which becomes, current prepares one of hydrogel most promising method.Enzymatic free radical
Polymerization belongs to one kind of enzyme-catalyzed polymerization, prepared hydrogel in this way in recent years and has gradually come into everybody visual field, this
Hydrogel made from method is very flexible in terms of regulation physics and chemical property, and with good enzyme response.But mesh
It is preceding not yet to have method simple and easy to apply, the nanogel for preparing excellent performance is reacted using enzyme-catalyzed polymerization.
The content of the invention
It is an object of the present invention to overcome the above-mentioned drawbacks of the prior art and provide a kind of nanogel is scattered
Property the good and enzymatic small molecule self assembly simple and easy to apply method for preparing nanogel.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of method that enzymatic small molecule self assembly prepares nanogel, this method is surface first successively to nano particle
Amination modification, carboxylated modification are carried out, is reacted afterwards by acid amides by enzyme modification in nano grain surface, is eventually adding small point
Sub- peptide, in the presence of enzyme, small-molecular peptides are converted into gelator, and are self-assembly of nanogel in nano grain surface.
The gelator newly formed is preferential in nano grain surface assembling, so that forming gel is wrapped in nano grain surface.
This method specifically includes following steps：
(1) the amination modification of nano particle：Nano particle is scattered in a solvent, amino silane coupling is added afterwards
Agent, and the nano particle that amination modification is obtained after 8-24h, washing is reacted at 40-70 DEG C；
(2) the carboxylated modification of nano particle：The nano particle that amination is modified is scattered in organic solvent, Zhi Houjia
Enter succinic anhydride, and stir the nano particle that carboxylated modification is obtained after 6-18h, washing；
(3) enzyme modification of nano particle：The nano particle that carboxylated is modified is dispersed in biological buffer, and adjusts pH
Value is to 5-6, after activated carboxyl, adds enzyme and obtained after acid amides reaction, washing the nano particle of enzyme modification；
(4) self assembly of nano grain surface small molecule prepares nanogel：The nano particle of enzyme modification is dispersed in solvent
In, and pH value is adjusted to 7-8, add afterwards after small-molecular peptides, stirring reaction 6-12h, that is, obtain described nanogel.
Described nano particle includes one or both of nano SiO 2 particle or ferroferric oxide nano granules.
Wherein, nano SiO 2 particle is usedPrepared by Hydrolyze method, the ratio of regulation heterogeneity can prepare difference directly
The nano SiO 2 particle in footpath.If from ferroferric oxide nano granules, when the ferroferric oxide nano granules of enzyme modification connect
When contacting small-molecular peptides, enzymic catalytic reaction occurs on the surface of ferroferric oxide nano granules, produced on this surface gel because
Son, as catalytic reaction proceeds, the gelator concentration increase on ferroferric oxide nano granules surface is carried out from group immediately
Dress forms one layer of hydrogel, prevents after gel layer formation enzyme to be reacted with small-molecular peptides, thus can only form nanogel and
Non- macroscopic hydrogel.
The particle diameter of described nano particle is 180-220nm.Preferably 200nm, 200nm size are conducive to follow-up life
Thing application, the diseased region such as being easier penetration barriers and reach tumor and cancer, for pharmaceutical carrier, due to what is passively transported
Effect, can realize medicine controlled releasing and targeted therapy.
In step (1) and step (4), described solvent includes one or both of water or ethanol；In step (2), institute
The organic solvent stated includes one or both of N,N-dimethylformamide or 1-METHYLPYRROLIDONE；It is described in step (3)
Biological buffer include one or both of MES or citric acid.
In step (1), described amino silicane coupling agent includes 3- aminopropyl triethoxysilanes or 3- aminopropyl front threes
One or both of TMOS；In step (3), described enzyme includes carboxy-lesterase or one kind in acid phosphatase or two
Kind.
In step (4), described small-molecular peptides include NapFFES or Fmoc-Tyr (H2PO3One or both of)-OH,
Described NapFFES chemical structural formula is shown below：
Described Fmoc-Tyr (H2PO3)-OH chemical structural formula is shown below：
In step (3), described activated carboxyl process is：NHS and EDC is added, 1.5-2.5h is stirred afterwards, often
The EDC described in NHS and 220-240mg being separately added into 100mL biological buffers described in 100-120mg.NHS is N- hydroxyl ambers
Amber acid imide, EDC is 1- (3- dimethylamino-propyls) -3- ethyl-carbodiimide hydrochlorides.
In acid amides course of reaction described in step (3), reaction temperature is 15-35 DEG C, and the reaction time is 5-7h.
In step (1), in every 100mL solvents, nano particle and the 0.8-0.9mL institute being separately added into described in 800-1200mg
The amino silicane coupling agent stated；
In step (2), per the nanometer for the amination modification in 100mL organic solvents, being separately added into described in 800-1200mg
Particle and the succinic anhydride described in 80-120mg；
In step (3), per the nanometer for the carboxylated modification in 100mL biological buffers, being separately added into described in 80-120mg
Particle and the enzyme described in 40-60mg；
In step (4), in every 100mL solvents, the nano particle and 15- of the enzyme modification being separately added into described in 80-120mg
Small-molecular peptides described in 25mg.
Compared with prior art, the invention has the characteristics that：
1) enter small-molecular peptides by enzymatic and be converted into gelator, and be preferentially assembled into small molecule in nano grain surface to coagulate
Glue, and then prepare nanogel, the nanogel has bioactivity and multifunctionality, can as growth factor, medicine and
The carrier of cell, the treatment repaired applied to cell culture, wound etc., new thinking is provided for the preparation of nanogel, it is rich
The rich preparation method of nanogel, and the present invention have it is gentle efficiently, the advantage such as biocompatibility is strong, greatly reduce all kinds of
The time of solvent cleaning and cost, are conducive to the biologic applications such as follow-up biological developing, HIFU ultrasounds；
2) by changing the concentration of small-molecular peptides, it can realize that the thickness of gel layer is controllable, with the increasing of gel layer thicknesses
Plus, corresponding change can occur for surface potential, and the system plastic is safe and efficient, can with good intensity and bioactivity
Applying in the field such as cell culture or organizational project；
3) crosslinking rate and plastic speed of nanogel can be regulated and controled by changing the concentration or consumption of each component, and can root
According to needing to realize gel in-situ, flexibility is good.
Brief description of the drawings
Fig. 1 is the preparation process schematic diagram of nanogel in embodiment 1；
Fig. 2 is the NapFFES prepared in embodiment 1 NMR spectra；
Fig. 3 is the SiO for preparing in embodiment 12The SEM spectrum of nano particle and nanogel；
Fig. 4 is the preparation process schematic diagram of nanogel in embodiment 2；
Fig. 5 is the Fe for preparing in embodiment 13O4The SEM spectrum of nano particle and nanogel.
Embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.The present embodiment is with technical solution of the present invention
Premised on implemented, give detailed embodiment and specific operating process, but protection scope of the present invention is not limited to
Following embodiments.
Embodiment 1：
A kind of enzymatic small molecule self assembly prepares the method for nanogel as shown in figure 1, specifically including following steps：
(1)SiO2The synthesis of nano particle：
147mL absolute ethyl alcohols, 2.5mL deionized waters, 7.5mL ammoniacal liquor are stirred at 40 DEG C, then added thereto
2.8mL teos hydrolysis 3h, are centrifuged afterwards, and are washed three times with absolute ethyl alcohol, obtain silicon ball, i.e. SiO2Nano particle.
(2) solid-phase synthesis prepares NapFF：
(2-1) weighs 0.3g (0.405mmol) 2- chlorine trityl chloride resin in synthesis in solid state device, and adds 2.5mL
DMF, is passed through nitrogen 5min afterwards, 2- chlorine trityl chloride resins is fully swelled；
(2-2) presses dichloromethane from the synthesis in solid state device equipped with 2- chlorine trityl chloride resins with nitrogen and removes clean；
314mg (0.81mmol) Fmoc amino acid protected is dissolved in 2mL DMF by (2-3), adds 145 μ L
(0.83mmol) DIEA (DIPEA), is then transferred into above-mentioned solid phase synthesizer, is passed through nitrogen, at room temperature
React 2h；
(2-4) adds 130 μ L methanol in synthesis in solid state device, reacts 5 minutes, and liquid in the synthesis in solid state device is pressed net,
Washed three times, each 30s, then washed with the DMF of 5mL percents by volume containing piperidines 20% with 2.5mL DMF, finally use 2.5mL
DMF is cleaned 3 times, each 30s；
(2-5) take second Fmoc protection amino acid 256mg (0.673mmol), 120 μ L (0.573mmol) DIEA and
Solution is made in 2.5mL dry DMFs, and the solution prepared is added in above-mentioned solid phase synthesizer, is passed through nitrogen reaction 2h；
(2-6) repeats (2-5) method and sequentially adds institute's amino acid in need, is then washed with DMF 3 times, then use hexamethylene
Alkane and isopropanol are respectively washed three times, each 1mL；
(2-7) presses the volume ratio 1 of trifluoroacetic acid and anhydrous methylene chloride:9, it is 10% to be configured to concentration of volume percent
Trifluoroacetic acid solution, cuts product from 2- chlorine trityl chloride resins, concentration, adds pH=3 watery hydrochloric acid to remove residual
Trifluoroacetic acid, drained with oil pump, obtain crude product, HPLC separating-purifyings are used afterwards, that is, be made NapFF, its chemical structural formula is such as
Shown in following formula：
(3) NapFFES synthesis：
480mg (1mmol) NapFF, 115mg (1mmol) NHS is dissolved in 20mL THF by (3-1), is added afterwards
230mg (1.2mmol) EDC, activates 2h, excessive monoethanolamine (2mL) reaction 6h is added, then concentrated by rotary evaporation, with pH=3's
Watery hydrochloric acid is washed three times, is freezed, is obtained solid；
(3-1) is reacted obtained solid and is dissolved in 20mL chloroforms by (3-2), addition 3mmol (0.5ml) DIEA (N,
N- diisopropylethylamine) and 3mmol (300mg) succinic anhydride, react a whole night, filtering takes its filtrate to rotate, dilute with pH=3
Salt acid elution three times, it is lyophilized to obtain crude product, HPLC separating-purifyings are used, that is, obtain NapFFES.
Fig. 2 is the NapFFES prepared NMR spectra, as seen from Figure 2, by above-mentioned steps, has synthesized small
Molecular peptide NapFFES.
(4) silicon ball surface amination is modified：The silicon ball that will be prepared in (1), is distributed in 120mL ethanol, at 40 DEG C
Stirring, and 1mL amino silicane coupling agent (3- aminopropyl triethoxysilanes) is added thereto, a whole night is reacted, ethanol is used
Washing is several times.
(5) silicon ball surface carboxylated is modified：It is distributed to after the amidized silicon ball prepared in (4) is washed with ethanol
In DMF, the DIEA (1mL) of three times and the succinic anhydride (100mg) of three times are added thereto, stir 6h, second is used after reaction completely
Alcohol is washed three times, then is washed with deionized three times, is freezed with standby.
(6) silicon ball surface modification esterase：The silicon ball of (5) carboxylated is distributed in MES 100mL, adjusted
PH, makes pH about 5.5 or so, adds 1mmol NHS (115mg), 1.2mmol EDC (230mg) activated carboxyl 2h, then adds
6h is stirred at room temperature in 50mg carboxy-lesterase, is washed with deionized afterwards three times, standby after freezing.
(7) prepared by silicon ball surface small molecule self-assembled nanometer gel：The silicon ball that 100mg has modified esterase is distributed to 100mL
In deionized water, regulation pH makes pH be approximately equal to 7.5, and the NapFFES that 20mg is added thereto reacts a whole night, under esterase effect
Presoma NapFFES is changed into gelator NapFFE, and nanogel is self-assembly of in silica surface.It is washed out surpassing
Sound, is re-dispersed into water and obtains nanogel, or lyophilized with standby.Wherein, NapFFES chemical structural formulas such as following formula institute
Show：
Fig. 3 is the SiO prepared2The SEM spectrum of nano particle and nanogel, as seen from Figure 4, is prepared
Nanogel in, SiO2Nano grain surface is wrapped with one layer of hydrogel layer.
Embodiment 2：
A kind of enzymatic small molecule self assembly prepares the method for nanogel as shown in figure 4, specifically including following steps：
(1)Fe3O4The synthesis of nano particle：
First by 0.54g FeCl3It is dissolved in (V in the mixed solution of 20mL ethylene glycol and diethylene glycolEG/VDEG=3/17),
Stir after 30min, add 2g polyvinylpyrrolidones (PVP, K30), be then heated to 120 DEG C, and be passed through N under agitation2, shape
Into clear solution, react after 1h, add 1.5g NaOAc, and stop heating, be stirred vigorously after 30min, solution is transferred to
In 50mL ptfe autoclaves, then reactor is placed in 200 DEG C of baking ovens keeps 12h.Room temperature is cooled to after the completion of reaction,
Take out reactor, obtained black product i.e. magnetic ferroferric oxide nanoparticle (MNPs), successively with ethanol and deionized water
Cleaning several times, is dispersed in standby in 20mL deionized waters afterwards.
(2) ferroso-ferric oxide (Fe3O4) nano particle surface modification
Take the Fe of the above-mentioned preparations of 2mL3O4In the mixed liquor for being dispersed in 80mL ethanol and 80mL deionized waters, ultrasonic disperse
After 30min, 6mL APTES (3- aminopropyl triethoxysilanes) are added, and 24h is reacted under 70 DEG C of heating stirrings, are led to simultaneously
Enter N2.After the completion of reaction, the particle several times, obtained is cleaned with ethanol and deionized water respectively and aoxidizes three for the four of surface amination
Iron, i.e. Fe3O4-APTES.Then by Fe3O4- APTES is dispersed in 100mL DMF, adds 10% succinic anhydride, stirring
18h is reacted, is cleaned afterwards with second alcohol and water, obtains the ferroso-ferric oxide Fe of surface carboxyl groups3O4- COOH, by Fe3O4- COOH points
It is dispersed in standby in 20mL deionized waters.
(3) ferroso-ferric oxide (Fe3O4) nano-hydrogel preparation
By Fe3O4After-COOH is modified with acid phosphatase (AP), the Fmoc-Tyr that 100mL concentration is 0.5mg/mL is scattered in
(H2PO3) in-OH solution, after ultrasonic disperse, certain time is stirred, Magneto separate removes unreacted material after the completion for the treatment of self assembly,
Three times are cleaned with deionized water again and obtains nanogel, nanogel is scattered standby in deionized water.
Ferriferrous oxide nano hydrogel removes Fmoc-Tyr in preparation process by the digestion effect of acid phosphatase
(H2PO3) phosphate radical in-OH, make small molecule Fmoc-Tyr (H2PO3)-OH is changed into Fmoc-Tyr-OH, is changed into thin from hydrophily
Aqueous, Fmoc-Tyr-OH is because hydrophobicity is in Fe3O4Particle surface self assembly, forms one layer of small molecule gel.
Fig. 5 is the Fe prepared3O4The SEM spectrum of nano particle and nanogel, as seen from Figure 5, is prepared
Nanogel in, Fe3O4Nano grain surface is wrapped with one layer of hydrogel layer.
Embodiment 3：
A kind of method that enzymatic small molecule self assembly prepares nanogel, this method is surface first successively to nano particle
Amination modification, carboxylated modification are carried out, is reacted afterwards by acid amides by enzyme modification in nano grain surface, is eventually adding small point
Sub- peptide, in the presence of enzyme, small-molecular peptides are converted into gelator, and are self-assembly of nanogel in nano grain surface.
This method specifically includes following steps：
(1) the amination modification of nano particle：Nano particle is scattered in a solvent, amino silane coupling is added afterwards
Agent, and the nano particle that amination modification is obtained after 24h, washing is reacted at 40 DEG C, wherein, per in 100mL solvents, add respectively
Enter 800mg nano particles and 0.9mL amino silicane coupling agents；
(2) the carboxylated modification of nano particle：The nano particle that amination is modified is scattered in organic solvent, Zhi Houjia
Enter succinic anhydride, and stir the nano particle that carboxylated modification is obtained after 12h, washing, wherein, per in 100mL organic solvents, divide
Not Jia Ru 1000mg aminations modification nano particle and 100mg succinic anhydrides；
(3) enzyme modification of nano particle：The nano particle that carboxylated is modified is dispersed in biological buffer, and adjusts pH
Value adds NHS and EDC afterwards to 6, and stirs 1.5h activated carboxyls, adds enzyme, and carries out acid amides reaction 5h at 35 DEG C, washes
The nano particle of enzyme modification is obtained after washing, wherein, per in 100mL biological buffers, it is separately added into 120mg NHS, 220mg
The nano particle and 40mg enzymes of EDC, 120mg carboxylated modification；
(4) self assembly of nano grain surface small molecule prepares nanogel：The nano particle of enzyme modification is dispersed in solvent
In, and pH value is adjusted to 7.5, add afterwards after small-molecular peptides, stirring reaction 9h, that is, obtain nanogel, wherein, per 100mL
In solvent, the nano particle and 20mg small-molecular peptides of 100mg enzyme modifications are separately added into.
Wherein, nano particle is nano SiO 2 particle, and the particle diameter of nano particle is 200nm.Step (1) and step
(4) in, solvent is water；In step (2), organic solvent is 1-METHYLPYRROLIDONE；In step (3), biological buffer is 2-
Quinoline ethyl sulfonic acid.In step (1), amino silicane coupling agent includes 3- aminopropyl triethoxysilanes and 3- aminopropyl trimethoxy silicon
Alkane；In step (3), enzyme is carboxy-lesterase.In step (4), small-molecular peptides are NapFFES, and NapFFES chemical structural formula is as follows
Shown in formula：
Embodiment 4：
A kind of method that enzymatic small molecule self assembly prepares nanogel, this method is surface first successively to nano particle
Amination modification, carboxylated modification are carried out, is reacted afterwards by acid amides by enzyme modification in nano grain surface, is eventually adding small point
Sub- peptide, in the presence of enzyme, small-molecular peptides are converted into gelator, and are self-assembly of nanogel in nano grain surface.
This method specifically includes following steps：
(1) the amination modification of nano particle：Nano particle is scattered in a solvent, amino silane coupling is added afterwards
Agent, and the nano particle that amination modification is obtained after 8h, washing is reacted at 70 DEG C, wherein, per in 100mL solvents, add respectively
Enter 1200mg nano particles and 0.8mL amino silicane coupling agents；
(2) the carboxylated modification of nano particle：The nano particle that amination is modified is scattered in organic solvent, Zhi Houjia
Enter succinic anhydride, and stir the nano particle that carboxylated modification is obtained after 18h, washing, wherein, per in 100mL organic solvents, divide
Not Jia Ru 800mg aminations modification nano particle and 120mg succinic anhydrides；
(3) enzyme modification of nano particle：The nano particle that carboxylated is modified is dispersed in biological buffer, and adjusts pH
Value adds NHS and EDC afterwards to 5, and stirs 2.5h activated carboxyls, adds enzyme, and carries out acid amides reaction 7h at 15 DEG C, washes
The nano particle of enzyme modification is obtained after washing, wherein, per in 100mL biological buffers, it is separately added into 100mg NHS, 240mg
The nano particle and 60mg enzymes of EDC, 80mg carboxylated modification；
(4) self assembly of nano grain surface small molecule prepares nanogel：The nano particle of enzyme modification is dispersed in solvent
In, and pH value is adjusted to 7, add afterwards after small-molecular peptides, stirring reaction 12h, that is, obtain nanogel, wherein, it is molten per 100mL
In agent, the nano particle and 25mg small-molecular peptides of 80mg enzyme modifications are separately added into.
Wherein, nano particle is ferroferric oxide nano granules, and the particle diameter of nano particle is 220nm.Step (1) and step
(4) in, solvent is ethanol；In step (2), organic solvent includes DMF and 1-METHYLPYRROLIDONE；Step
(3) in, biological buffer includes MES and citric acid.In step (1), amino silicane coupling agent is 3- aminopropyls three
Methoxy silane；In step (3), enzyme is acid phosphatase.In step (4), small-molecular peptides are Fmoc-Tyr (H2PO3)-OH。
Embodiment 5：
A kind of method that enzymatic small molecule self assembly prepares nanogel, this method is surface first successively to nano particle
Amination modification, carboxylated modification are carried out, is reacted afterwards by acid amides by enzyme modification in nano grain surface, is eventually adding small point
Sub- peptide, in the presence of enzyme, small-molecular peptides are converted into gelator, and are self-assembly of nanogel in nano grain surface.
This method specifically includes following steps：
(1) the amination modification of nano particle：Nano particle is scattered in a solvent, amino silane coupling is added afterwards
Agent, and the nano particle that amination modification is obtained after 16h, washing is reacted at 55 DEG C, wherein, per in 100mL solvents, add respectively
Enter 1000mg nano particles and 0.85mL amino silicane coupling agents；
(2) the carboxylated modification of nano particle：The nano particle that amination is modified is scattered in organic solvent, Zhi Houjia
Enter succinic anhydride, and stir the nano particle that carboxylated modification is obtained after 6h, washing, wherein, per in 100mL organic solvents, divide
Not Jia Ru 1200mg aminations modification nano particle and 80mg succinic anhydrides；
(3) enzyme modification of nano particle：The nano particle that carboxylated is modified is dispersed in biological buffer, and adjusts pH
Value adds NHS and EDC afterwards to 5.5, and stirs 2h activated carboxyls, adds enzyme, and carries out acid amides reaction 6h at 20 DEG C, washes
The nano particle of enzyme modification is obtained after washing, wherein, per in 100mL biological buffers, it is separately added into 110mg NHS, 230mg
The nano particle and 50mg enzymes of EDC, 100mg carboxylated modification；
(4) self assembly of nano grain surface small molecule prepares nanogel：The nano particle of enzyme modification is dispersed in solvent
In, and pH value is adjusted to 8, add afterwards after small-molecular peptides, stirring reaction 6h, that is, obtain nanogel, wherein, it is molten per 100mL
In agent, the nano particle and 15mg small-molecular peptides of 120mg enzyme modifications are separately added into.
Wherein, nano particle includes nano SiO 2 particle and ferroferric oxide nano granules, the particle diameter of nano particle
For 180nm.In step (1) and step (4), solvent includes water and ethanol；In step (2), organic solvent is N, N- dimethyl methyls
Acid amides；In step (3), biological buffer is citric acid.In step (1), amino silicane coupling agent is 3- aminopropyl-triethoxies
Silane；In step (3), enzyme includes carboxy-lesterase and acid phosphatase.In step (4), small-molecular peptides include NapFFES and
Fmoc-Tyr(H2PO3)-OH, NapFFES chemical structural formula is shown below：
The above-mentioned description to embodiment is understood that for ease of those skilled in the art and using invention.
Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without passing through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel are according to the announcement of the present invention, and not departing from improvement and modification that scope made all should be the present invention's
Within protection domain.
Claims (10)
1. a kind of method that enzymatic small molecule self assembly prepares nanogel, it is characterised in that this method is first successively to nanometer
The surface of particle carries out amination modification, carboxylated modification, afterwards by acid amides reaction by enzyme modification in nano grain surface, most
After add small-molecular peptides, in the presence of enzyme, small-molecular peptides are converted into gelator, and are self-assembly of in nano grain surface
Nanogel.
2. the method that a kind of enzymatic small molecule self assembly according to claim 1 prepares nanogel, it is characterised in that should
Method specifically includes following steps：
(1) the amination modification of nano particle：Nano particle is disperseed in a solvent, amino silicane coupling agent to be added afterwards, and
The nano particle that amination modification is obtained after 8-24h, washing is reacted at 40-70 DEG C；
(2) the carboxylated modification of nano particle：The nano particle that amination is modified is scattered in organic solvent, and fourth is added afterwards
Dicarboxylic anhydride, and stir the nano particle that carboxylated modification is obtained after 6-18h, washing；
(3) enzyme modification of nano particle：The nano particle that carboxylated is modified is dispersed in biological buffer, and adjust pH value to
After 5-6, activated carboxyl, add enzyme and obtained after acid amides reaction, washing the nano particle of enzyme modification；
(4) self assembly of nano grain surface small molecule prepares nanogel：The nano particle of enzyme modification is scattered in a solvent, and
PH value is adjusted to 7-8, is added afterwards after small-molecular peptides, stirring reaction 6-12h, that is, obtains described nanogel.
3. the method that a kind of enzymatic small molecule self assembly according to claim 2 prepares nanogel, it is characterised in that institute
The nano particle stated includes one or both of nano SiO 2 particle or ferroferric oxide nano granules.
4. the method that a kind of enzymatic small molecule self assembly according to claim 3 prepares nanogel, it is characterised in that institute
The particle diameter for the nano particle stated is 180-220nm.
5. the method that a kind of enzymatic small molecule self assembly according to claim 2 prepares nanogel, it is characterised in that step
Suddenly in (1) and step (4), described solvent includes one or both of water or ethanol；In step (2), described is organic molten
Agent includes one or both of N,N-dimethylformamide or 1-METHYLPYRROLIDONE；In step (3), described biological buffer
Liquid includes one or both of MES or citric acid.
6. the method that a kind of enzymatic small molecule self assembly according to claim 2 prepares nanogel, it is characterised in that step
Suddenly in (1), described amino silicane coupling agent is included in 3- aminopropyl triethoxysilanes or 3- aminopropyl trimethoxysilanes
One or two；In step (3), described enzyme includes one or both of carboxy-lesterase or acid phosphatase.
7. the method that a kind of enzymatic small molecule self assembly according to claim 2 prepares nanogel, it is characterised in that step
Suddenly in (4), described small-molecular peptides include NapFFES or Fmoc-Tyr (H2PO3One or both of)-OH, it is described
NapFFES chemical structural formula is shown below：
8. the method that a kind of enzymatic small molecule self assembly according to claim 2 prepares nanogel, it is characterised in that step
Suddenly in (3), described activated carboxyl process is：NHS and EDC is added, 1.5-2.5h is stirred afterwards, biology is slow per 100mL
The EDC described in NHS and 220-240mg being separately added into fliud flushing described in 100-120mg.
9. the method that a kind of enzymatic small molecule self assembly according to claim 2 prepares nanogel, it is characterised in that step
Suddenly in the acid amides course of reaction described in (3), reaction temperature is 15-35 DEG C, and the reaction time is 5-7h.
10. the method that a kind of enzymatic small molecule self assembly according to claim 2 prepares nanogel, it is characterised in that
In step (1), in every 100mL solvents, described in the nano particle and 0.8-0.9mL being separately added into described in 800-1200mg
Amino silicane coupling agent；
In step (2), per the nano particle for the amination modification in 100mL organic solvents, being separately added into described in 800-1200mg
And the succinic anhydride described in 80-120mg；
In step (3), per the nano particle for the carboxylated modification in 100mL biological buffers, being separately added into described in 80-120mg
And the enzyme described in 40-60mg；
In step (4), in every 100mL solvents, nano particle and the 15-25mg institute for the enzyme modification being separately added into described in 80-120mg
The small-molecular peptides stated.
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